Carcinoma of unknown primary: Molecular tumor board‐based therapy
Authors: Aditya V. Shreenivas, Shumei Kato, Jingjing Hu, Catherine Skefos, Jason Sicklick, Razelle Kurzrock
Journal: CA A Cancer Journal for Clinicians (2022)

A 36-year-old woman presented to an outside hospital with abdominal pain in July of 2021. A computerized tomography (CT) scan of the abdomen and pelvis showed multiple, hypodense lesions within the left hepatic lobe that were concerning for metastatic disease. The largest liver lesion (3.1 × 2.2 cm) was biopsied, and pathology was consistent with metastatic, moderately differentiated adenocarcinoma. Immunohistochemistry (IHC) stains performed for further characterization of the tumor were positive for cytokeratin 7 (CK7) and CDX2 and negative for CK20, HepPar-1, Napsin A, and GATA-3, consistent with adenocarcinoma. The differential diagnosis included primary cholangiocarcinoma versus metastatic carcinoma of upper gastroesophageal or pancreaticobiliary origin. Of note, it was also suspected that the patient had amoebiasis, and she was on treatment with antibiotics. She later transferred her care to our university hospital. A follow-up CT scan of the abdomen and pelvis performed a few weeks after the initial presentation demonstrated hypodense liver lesions involving the right and left lobes as well as peripancreatic and gastrohepatic lymph nodes, which were suspicious for malignancy. Because the liver biopsy could not identify the primary tumor conclusively, subsequent endoscopic ultrasound and fine-needle aspiration of the peripancreatic lymph node were performed almost a month after the initial presentation. The cell block material obtained from fine-needle aspiration of the peripancreatic lymph node demonstrated cellular material with numerous groups of epithelioid tumor cells forming cribriform glands with background lymphoid tissue (Figure 1A). The epithelioid tumor cells showed enlarged and stratified nuclei, loss of polarity, and irregular nuclear membranes. The cribriform glands demonstrated luminal necrosis and frequent mitosis (Figure 1B). Morphologic findings were consistent with metastatic adenocarcinoma in a lymph node. A panel of IHC studies was performed to further categorize the tumor origin. The tumor cells were strongly positive for CK7 (Figure 1C), patchy positive for CDX2 (Figure 1D), and negative for GATA3, CK20, and HER2. The profile was consistent with upper gastrointestinal cancers, including gastric, esophageal, and pancreatobiliary primaries. Although IHC can be helpful in subtyping upper gastrointestinal versus lower gastrointestinal tumors, it is not very specific in distinguishing gastroesophageal adenocarcinomas from pancreatobiliary adenocarcinomas. Morphologically, there were no definitive features to distinguish the origin from gastric, esophageal, or pancreatobiliary sites. Some soft features that may suggest pancreaticobiliary origin are eosinophilic cytoplasm or foamy cytoplasm of the tumor cells. IHC is not helpful because CDX2 is a general marker for a gastrointestinal primary. The most popular classification of gastric cancer is the Lauren classification, with intestinal and diffuse subtypes.1 The intestinal subtype shows tubular and glandular elements, whereas the diffuse subtype displays noncohesive single tumors or signet ring-type cells without gland formation. In our case, the tumor cells demonstrated intestinal-type morphology. CT of the chest performed later to complete staging workup showed numerous lung nodules measuring up to 8 mm in size, consistent with metastatic pulmonary disease, and multiple, enlarged, pericardial/juxtaphrenic, paratracheal lymph nodes measuring approximately10 mm in size. The baseline level of the tumor marker carbohydrate antigen 19–9 (CA 19–9) was 2622 units/mL (reference, <37 units/mL), which increased to 3980 units/mL just before the patient started treatment, and the carcinoembryonic antigen level was 1.1 ng/mL (reference, <5 ng/mL). The lactate dehydrogenase (LDH) level was not checked at the time of presentation. The primary tumor could not be identified despite extensive workup; hence, it was labeled as carcinoma of unknown primary (CUP). However, the presence of elevated CA 19–9 levels was suggestive of metastatic adenocarcinoma of pancreatobiliary origin (although CA 19–9 can also be elevated in a variety of other malignancies).2 The patient was later seen by a medical oncologist in the clinic. Next-generation sequencing (NGS) of her tumor tissue and blood was ordered. Programmed death-ligand 1 (PD-L1) IHC expression was evaluated on liver tissue (pharm Dx22C3 antibody; Tempus platform) with a combined positive score (CPS) of 2. Unfortunately, the tissue sample was inadequate for NGS; however, liquid (blood) biopsy for NGS of circulating tumor DNA (ctDNA) could be performed. Blood-based NGS (311 genes; Foundation Medicine) demonstrated microsatellite-stable disease, FGFR2::LGSN fusion (variant allele frequency [VAF], 30.8%), FGFR2 rearrangement of intron 17 (VAF, 4.5%), FGFR2 amplification, and MYC amplification. Blood tumor mutational burden (TMB) was 4 mutations per megabase. Additional genomic profiling studies were done on her blood sample through the Guardant360 platform (approximately 73 genes evaluated; Guardant Inc.) confirmed microsatellite-stable disease and showed FGFR2 amplification (medium [++] amplification, magnitude between the 50th and 90th percentiles), MYC amplification (high [+++] amplification, >90th percentile), and a TMB of 5.74 mutations per megabase. IHC and NGS data for this case are clearly illustrated in Table 1. Foundation medicine liquid Cdx (311 gene panel) blood ctDNA NGS Germline testing 156 gene (Invitae customized cancer panel) on a blood sample Positive CK 7 ++ CDX2 + Negative CK20 HepPar-1 NapsinA GATA-3 Positive CK7 ++ CDX2 + Negative GATA3 CK20 HER2 PDL1 IHC (22C3 pharm Dx assay): CPS score 2 Tissue sample insufficient for genomic profiling MSI high not detected FGFR2::LGSN fusion VAF 30.8% FGFR2 rearrangement of intron 17 VAF 4.5% FGFR2 amplification MYC amplification bTMB:4 mut/Mb FGFR2 amplification +++ MYC amplification ++ bTMB:5.74 mut/Mb Targetable somatic alterations: FGFR-LGSN fusion and other FGFR2 alterations It is important to note that Guardant 360 Cdx (73-gene) assay does not report FGFR2 fusion, so we ordered a more comprehensive Foundation One (311-gene) liquid biopsy panel. Discordant results in NGS assays are common and can be explained by tumor heterogeneity and differences in methodology of testing. It is important for clinicians to be aware of certain limitations of commonly used NGS assays and to account for them before finalizing a personalized therapy plan for their patients. We also discussed our patient with our multidisciplinary Molecular Tumor Board members. The Molecular Tumor Board includes basic, translational, and clinical investigators, bioinformaticians, geneticists, pathologists, radiologists, and surgeons. The Molecular Tumor Board experts comprehensively reviewed the tumor's molecular characteristics, and the consensus was to proceed with systemic therapy. Real-world data from our earlier studies have shown that N-of-one strategies implemented based on the recommendation of a specialized molecular tumor board can lead to improved patient-related outcomes, including in treatment-naive patients with advanced disease.3-6 There is often a limited role for surgical intervention in the setting of multifocal, metastatic adenocarcinomas of upper gastrointestinal tract or hepatopancreatobiliary origin, which generally have poor prognoses. Surgical decision making is generally guided by three principles, namely, patient-related factors (e.g., comorbidities), tumor biology, and technical aspects of surgical resection. Methods to assess patient-related factors include the Charlson Comorbidity Index7 and the Eastern Cooperative Oncology Group performance score.8 Many of these scores will overlap in unfit patients, who often have multiple comorbidities as well as malnutrition, sarcopenia, and other physical and/or psychological determinants of poor candidacy for surgical resection. However, if a patient is a fit individual, the disease biology and surgical resectability are critical aspects to consider. In the current case, the patient is an otherwise healthy 36-year-old woman without past medical history who presented with multiple liver lesions involving the right and left hepatic lobes. She also had peripancreatic and gastrohepatic lymphadenopathy. Therefore, it is crucial to reason that this is bad disease biology because she has presented with multifocal, synchronous nodal and hepatic metastatic disease. Although this aspect of the metastatic disease is technically resectable via an extended right hepatectomy with lymphadenectomy, this would not be recommended because this approach does not account for the lung nodules and the very high likelihood of widespread microscopic disease. Furthermore, we know that the surgical intervention itself, anesthetics, and transfusions are immunosuppressive and may be a source of enhancing disease progression postoperatively. This patient needs systemic therapy. Moreover, should complications arise from an aggressive surgical intervention, there is the added potential risk of further delaying the initiation of systemic therapy. The surgical oncologist Blake Cady once described that the technical details of surgical procedures (the princes and princesses of the realm) frequently try to overthrow the powerful forces of biology (the king) and the selection of cases (the queen), usually with no long-term success but with some temporary victories.9 In other words, biology trumps surgical techniques, even in the most skilled of hands. In general, surgical intervention in this case should only be considered for urgent/emergent issues, such as obstruction, bleeding, or perforation. However, should significant disease reduction be achieved via systemic therapy, then it may be reasonable to consider resection of residual disease if the potential for benefiting the patient should outweigh the risk of complications or death from the operation. Taking into consideration the advanced-stage, aggressive nature and lack of highly effective conventional systemic therapy options for patients who have CUP, with a median survival <1 year,10 it was reasonable to offer this patient treatment with matched therapies. Combination therapy with the FGFR inhibitor pemigatinib (for the FGFR alterations; 13.5 mg orally; 2 weeks on, 1 week off) and pembrolizumab (for the PD-L1 positivity; 200 mg intravenously every 3 weeks) was started. She consented to participate in the PREDICT (Profile Related Evidence Determining Individualized Cancer Therapy) study (ClinicalTrials.gov identifier NCT02478931). After 2.5 months of treatment, imaging studies showed a significant treatment response, with decreased pulmonary, thoracic, liver, and lymph node disease. Follow-up imaging studies after 5 months of treatment showed the complete resolution of index lesions in segment IVB of the liver and a significant reduction in the size of other liver lesions (Figure 2). In addition, a significant biochemical response was also observed; the CA 19–9 level dropped from 3980 units/mL pretherapy to 70 units/mL after 2 months of therapy and, more recently, to 30 units/mL (a normal level; Figure 3) Overall, the patient had a sustained partial response ongoing for almost 7 months at the time we were preparing this article. She has tolerated therapy well. CUP is a heterogeneous group of cancers, accounting for approximately 3%–5% of malignancies, and is defined by the presence of metastatic disease with no identified primary tumor at the time of clinical presentation.11 CUPs are categorized by pathology into five categories according to European Society of Medical Oncology clinical practice guidelines: well differentiated and moderately differentiated adenocarcinomas, poorly differentiated carcinomas (including poorly differentiated adenocarcinomas), squamous cell carcinomas, carcinomas with neuroendocrine differentiation, and undifferentiated neoplasms.12 International consensus guidelines also recommend the meticulous application of IHC to identify the tissue of origin and exclude potentially chemosensitive and curable tumors.12, 13 CUP carries a dismal prognosis. The median overall survival of unfavorable CUP ranges from 6 to 12 months.14 Some studies have also described two prognostic subsets of CUPs according to their performance status and serum levels. Patients with good performance status and normal LDH have a median overall survival of approximately 1 year compared with approximately 4 months for those with elevated LDH or poor performance status.15 A population-based study showed that adenocarcinoma was the most common histology (33.4%) among CUPs when metastases were limited to lymph nodes and had a median survival of approximately 8 months.11 Only approximately 15%–20% of these cancers carry a favorable prognosis.12 There is still debate regarding therapeutic options for patients with CUP because traditional treatment approaches for cancer rely on the site of origin. CUP, therefore, remains an area of unmet need. Our patient had moderately differential adenocarcinoma with a likely upper gastrointestinal primary. The most common upper gastrointestinal adenocarcinomas include gastric, esophageal, and pancreatobiliary primaries. Gastric cancer is the fifteenth most common cancer by incidence, with 27,000 new cases diagnosed in 2019, representing approximately 2% of all new cancers.16 Gastric adenocarcinoma has predisposing risk factors, including Helicobacter pylori infection and genetic abnormalities. Tumor suppressor genes, including TP53, CDKN2A, and APC, and the TGF-β, MET, and HER2 oncogenes are frequently aberrant in gastric adenocarcinomas.17 Esophageal cancer is the eighteenth most common malignancy in the United States. The majority of esophageal adenocarcinomas develop in association with Barret esophagus. Inactivation of the tumor suppressor proteins p16, p27, and p53 and the APC gene and overexpression of cyclin-D1 are the most common molecular alterations in these tumors.18 Pancreatic cancer is an aggressive and catastrophic disease because of rapid progression, presentation at an advanced stage, and a tendency to be resistant to adjuvant chemoradiation/surgical therapy. It has a poor 5-year survival rate of 2%–9% and remains a leading cause of cancer-related mortality in the United States.19 Pancreas cancers are often associated with molecular alterations in the KRAS, TP53, CDKN2A/CDKN2B, BRCA, and SMAD4 genes.20 Cholangiocarcinoma is an aggressive hepatobiliary neoplasm arising from bile ducts in the liver. There are three main types of cholangiocarcinoma: intrahepatic, perihilar, and extrahepatic. Intrahepatic cholangiocarcinoma (iCCA) is the second most common primary hepatic malignancy next to hepatocellular carcinoma. iCCA has two main subtypes: large duct and small duct. Large-duct iCCA arises near the hepatic hilus, and small-duct iCCA occurs in the periphery. They show a ductal or tubular pattern with fibrous stroma. Molecular alterations in the KRAS, TP53, IDH1/IDH2, ARID1A, EGFR, HER2, and FGFR2 genes are common in these cancers.21 The absence of a KRAS mutation and the presence of an FGFR2 fusion on comprehensive genomic profiling seen in this case was consistent with the genetic profile of cholangiocarcinoma. However, this evidence was, not enough to rule out pancreatic adenocarcinoma because approximately 10% of pancreatic adenocarcinomas are KRAS wild type and are sometimes enriched with FGFR2 fusions.22 In addition, according to some other estimates, FGFR2 fusions can be found in approximately 1.1% of all cancers, including pancreatic adenocarcinomas, gastric adenocarcinomas, and cholangiocarcinomas.23 Despite multimodality diagnostic imaging procedures and an extensive review of histology and IHC stains of tumor, the tissue of origin could not be determined in this case, so we based our treatment on NGS results. According to some estimates, CUPs harbor a median of four deleterious genomic alterations per tumor,24 and at least 60%–65% of patients with CUPs can be eligible for molecular-guided therapy.25 Furthermore, in a study of >1700 patients with CUP, almost 50% had high levels of evidence alterations (level 1, level 2, or resistance 1; OncoKB) when only blood-derived ctDNA was analyzed.26 Earlier clinical studies from our group also demonstrated that both tissue-based and blood-based NGS tests reveal potentially effective therapeutic options for these patients (although the trials were not randomized).4, 25, 26 The efficacy and safety of NGS-informed, customized combination therapy was reported in the analysis of the I-PREDICT family studies, including in treatment-naive patients with a variety of lethal cancers such as CUP.5, 6, 26 The studies identified and matched the common pathognomonic molecular alterations with their respective gene-targeted and immune-targeted therapies. The strategy resulted in significantly improved disease control rates (stable disease ≥6 months/partial response/complete response, 50% vs. 22.4%; p = .028) and longer median progression-free survival rates (6.5 vs. 3.1 months; p = .001) and overall survival rates (not reached vs. 10.2 months; p < .048) in heavily pretreated patients with advanced malignancies who received matched therapy that affected more than one half of their genomic/immunomic alterations versus those who received treatment with lesser degrees of matching. An ongoing randomized phase 2/3 study is evaluating the efficacy of matched NGS-based therapies versus standard-of-care, platinum-based chemotherapies in patients newly diagnosed with unfavorable CUP (ClinicalTrials.gov identifier NCT03498521).27 FIGHT-202 was a multicenter, open-label, single-arm study of pemigatinib that enrolled patients with cholangiocarcinoma who had disease progression after at least one prior treatment and had documented FGF/FGFR gene status (ClinicalTrials.gov identifier NCT02924376). The study demonstrated a response rate of 35.5% (median duration of response, 9.1 months) in patients with FGFR2 gene rearrangements/fusions.28 The biomarkers most commonly associated with response to immunotherapy include PD-L1, tumor-infiltrating lymphocytes, microsatellite instability, mismatch repair, and TMB. The combined positive score (CPS) is an important tool for measuring PD-L1 expression. The CPS equals the number of PD-L1–positive tumor cells and lymphocytes, divided by the total number of viable tumor cells, multiplied by 100. Earlier studies have shown that cholangiocarcinomas and other gastrointestinal cancers with positive PD-L1 expression (CPS ≥1%) can respond to immunotherapy.29, 30 Traditional tissue biopsies have remained the gold standard for obtaining molecular information from tumors. However, clinicians often encounter situations in which inaccessibility of the tumor for routine tissue biopsy procedures or inadequate quantity or quality of a tissue sample for genomic profiling (as in our patient) results in the lack of molecular information, restricting the treatment options. This is an especially challenging situation for patients with CUP tumors, in whom the location of primary cancer is not known and standard systemic therapies are limited. Earlier studies have demonstrated that liquid biopsies using circulating tumor cells and ctDNA derived from the blood of patients with cancer can be a useful source of molecular information when tumor tissue is not available.31, 32 Circulating tumor cells have also been used to identify the primary tumor location in CUP using an algorithmic multiplex antibody set, which applies conjugated antibodies specific to CK7, CK20, and other common diagnostic proteins.31 Studies have also demonstrated that low-coverage, genome-wide sequencing of cell-free DNA from patients with cancer can be exploited to obtain relevant molecular information for situations in which tissue biopsy is not feasible.33 In one of our recent studies, genomic alterations (including variants of unknown significance) were found in approximately 90% of blood-derived cell-free DNA samples from evaluable patients who had CUP (N = 1931).26 The median number of deleterious molecular alterations was four. Furthermore, we also evaluated patients who had CUPs for outcome and found that those who received therapies with high degrees of matching had longer median progression-free survival (10.4 vs. 2.5 months; p = .002) and overall survival (13.4 vs. 5.7 months; p for trend = .07) and had a higher clinical benefit rate (stable disease ≥6 months/partial response/complete response, 83% vs. 25%; p = .003) than those who received unmatched drugs or drugs with low degrees of matching26; a high degree of matching often required a tailored combination of drugs matched to as many alterations as possible. Other studies24, 25 have also reported findings in support of effectively using liquid biopsy to gather molecular information for formulating an individualized treatment plan for these patients. These blood-based studies are consistent with other reports interrogating tissue NGS and showing that the greater the number of pathogenic alterations targeted, the better the outcome; that targeting multiple alterations often requires individualized combination therapy; and that it is the degree of matching, but not the number of drugs given, that predicts response in multivariable analysis.6 The fibroblast growth factor (FGF)/FGFR family consists of >20 FGF ligands, and these glycoproteins bind to FGFRs.34 There are five distinct FGFR receptors. FGFR1–FGFR4 are encoded respectively by the FGFR1–FGFR4 genes, and FGFR5 is encoded by FGFRL1 genes.35 FGFR1–FGFR4 are structurally comprised of extracellular immunoglobulin domains I–III, a transmembrane hydrophobic segment, and an intracellular tyrosine kinase domain. Interestingly, FGFR5 does not have an intracellular tyrosine kinase domain and lacks receptor tyrosine kinase activity.36 FGFRs are involved in several physiologic processes: embryonic development, cell cycle regulation, phosphorous, vitamin D and bile acid homeostasis, blood vessel formation, and wound healing.35 Aberrations in FGF/FGFR signaling can also lead to tumorigenesis. Molecular alterations in FGFR1–FGFR4, including gene rearrangements or fusions, single-nucleotide variants, and copy number amplifications, have been reported in 5%–10% of all cancers.37 Activation of the FGF/FGFR pathway can stimulate downstream intracellular cascades by the phosphorylating phosphoinositide 3 kinase/protein kinase (PI3/AKT), mitogen-activated protein kinase (MAPK), and signal transducer and activator of transcription (STAT) pathways. The RAS-MAPK and PI3K-AKT pathways are stimulated by the phosphorylation of FGFR substrate 2.35 Phosphorylated FGFR substrate 2α binds the membrane-anchored adaptor protein growth factor receptor-bound 2 (GRB2) and the tyrosine phosphatase Src homology-2 domain-containing phosphatase 2 (SHP2).38 GRB2 further activates the RAS-MAPK pathway through the recruitment of guanine nucleotide exchange factor son of sevenless (SOS) and the PI3K-AKT pathway through the recruitment of GRB2-associated binder 1 (GAB1) to the signaling complex.35 In addition, phosphorylated FGFR kinase also cross-talks with and activates the enzyme phospholipase C γ (PLC-γ), which produces inositol triphosphate 3 and diacyl glycerol by the hydrolysis of phosphatidylinositol 4,5-bisphosphate (PIP2), and induces calcium ion release from intracellular stores and the activation of downstream signaling pathways. Diacyl glycerol activates the enzyme protein kinase C and its downstream signaling pathways.35 All of these processes ultimately lead to tumor cell proliferation, differentiation, angiogenesis, and anti-apoptosis. Additional details are included in (Figure 4). NGS performed on the blood sample-derived ctDNA from our patient with CUP revealed a FGFR2::LGSN fusion (VAF, 30.8%), an FGFR2 rearrangement of intron 17 with a VAF of 4.5%, and FGFR2 amplifications (Figure 2). The above findings encouraged us to offer this patient anti-FGFR–based therapy. It is important to note that both FGFR2::LGSN fusion and FGFR2 rearrangement of intron 17 have been reported by investigators in cholangiocarcinoma39; hence, we decided to use an FGFR2-selective inhibitor approved in cholangiocarcinoma. Pemigatinib is a potent, small-molecule, selective tyrosine kinase inhibitor that blocks the intracellular tyrosine kinase activity of FGFR1–FGFR4.40 On the basis of results from the FIGHT-202 study,28 pemigatinib has recently been granted US Food and Drug Administration (FDA) approval for patients with previously treated, unresectable, locally advanced or metastatic cholangiocarcinomas that harbor FGFR2 fusions or rearrangements.28 Other selective FGFR inhibitors, such as erdafitinib41 and infigratinib,42 have also received FDA approval in the management of previously treated, unresectable, locally advanced or metastatic urothelial carcinoma (FGFR2/FGFR3 alterations) and cholangiocarcinoma (FGFR2 fusions), respectively. Agents such as derazantinib,43 futibatinib,44 and others are in various stages of drug development We have included some examples of published studies of selective FGFR inhibitors in Table 2.26, 38-41, 45 Apart from pemigatinib, infigratinib is the only other FDA-approved, selective FGFR2 inhibitor for cholangiocarcinoma. We chose pemigatinib because we had more clinical experience using it in similar clinical settings, and it was FDA approved almost a year before infigratinib and was easily accessible at the time of treatment initiation. Pemigatinib FDA approval in iCCA 4/17/2020 FGFR1: 0.4nm FGFR2: 0.5nm FGFR3: 1nm FGF4: 30nm FIGHT-20228 (N=103) ORR - 35.5% mPFS 6.9mths mDOR – 7.5 months Hyperphosphatemia (~60%/0%) Diarrhea (~47%/3%) Fatigue (~43%/5%) Alopecia (~49%/0%) Dysgeusia (~40%/0%) Nausea (~40%/2%) Stomatitis (~29%/5%) Dry Eye (~26%, 1%) Hypophosphatemia (~23%/12%) Infigratinib FDA approval in iCCA 5/28/2021 FGFR1: 0.9nm FGFR2: 1.4nm FGFR3: 1nm FGF4: 60 nm CBGJ398X220442 Phase II ORR- 23% mDOR- 5 months mPFS -7.3mths mOS-12.2 mths Hyperphosphatemia (~90%/13%) Stomatitis (~56%/15%) Diarrhea (~24%/3%) Dry Eye (~44%, 0%) Fatigue (~44%/4%) Alopecia (~38%/0%) Dysgeusia (~32%/0%) Dry Mouth (~25%/0%) Adverse reaction percentages verified from Infigratinib package insert Erdafitinib FDA approval in bladder ca 4/12/2019 FGFR1: 1.2nm FGFR2: 2.5nm FGFR3: 3nm FGF4: 5.7 nm BLC200141 Phase II ORR- 40% mOS -13.8 mths mPFS -5.5 mths Hyperphosphatemia (~77%/2%) Stomatitis (~58%/10%) Diarrhea (~51%/4%) Dry mouth (~46%/0%) Fatigue (~32%/2%) Dysgeusia (~37%/1%) Dry eye (~19%/1%) Paronychia (~17%/3%) Blurred vision (~17%/0%) Futibatinib FGFR1: 3.9nm FGFR2: 1.3nm FGFR3: 1.6nm FGF4: 8.3 nm FOENIX44CCA2 Phase II ORR – 41% mDOR 9.5mths mPFS 8.9 mths mOS 20mths Hyperphosphatemia (~85%/30%) Alopecia (~33%/ N/A%) Diarrhea (~28%, N/A%) Dry mouth (~30.0%, N/A%) Fatigue (~25%/N/A%) Dry Skin (~27%/N/A%) Derazantinib FGFR1: 4.5nm FGFR2: 1.8nm FGFR3: 4.5nm FGF4: 34 nm CSF1R VEGFR2 Phase I/II Study (N=29)43 ORR 20.7% mPFS 5.7 mths Hyperphosphatemia (~76%/10%) Dry mouth (~44.8%/0%) Nausea (~44.8%/0%) Asthenia/Fatigue (~69%/10%) Stomatitis (~7%/3%) Alopecia (~24%/0%) Dry Eye(~17.2%/3.4%) The patient's tumor tissue demonstrated low positive PD-L1 expression (CPS = 2) by IHC, prompting the addition of the checkpoint inhibitor (anti–PD-1) pembrolizumab to the FGFR inhibitor (pemigatinib)-based therapy. Preclinical studies have also shown that anti–PD-1 agents work synergistically with FGFR inhibitors to enhance their tumoricidal effect by antitumor immune activation. Combining these two therapies leads to decreased expression of PD-1, expansion of T-cell clones, and alteration of the tumor microenvironment by immunologic changes mediated by the FGFR inhibitor.46 A combination of anti–PD-1 agents and FGFR inhibitors also is active in the clinic. For instance, the FIERCE-22 trial showed that vofatamab, a selective FGFR3 inhibitor, in combination with pembrolizumab in metastatic urothelial carcinoma was associated with responses in one third of patients regardless of FGFR alteration status (ClinicalTrials.gov identifier NCT03123055).47 A recently published phase 2 trial assessed pembrolizumab in a broad range of unresectable or metastatic rare tumors, included a cohort for CUP, and reported an objective response rate of 23% in the patients with CUP.48 Furthermore, combination immunotherapies, such as nivolumab (anti–PD-1) and ipilimumab (anti-CTLA4), also have activity in advanced biliary tract cancers; Klein et al. reported a response rate of 23% with nivolumab and ipilimumab in 39 patients with heavily pretreated, microsatellite-stable cholangiocarcinomas and gallbladder cancers.49 The above-mentioned findings have encouraged investigators to evaluate efficacy of other FGFR inhibitors in combination with immune checkpoint inhibitors in various tumor types. Examples of ongoing trials of FGFR inhibitors and combination therapies are listed in Table 3. Phase II match study (NCT02465060) Phase II single-arm study (NCT05174650) Phase II study (NCT04003610) FIGHT-205 Three groups: Pemigatinib + Pembrolizumab Pemigatinib alone Standard of care (gemcitabine + carboplatin or Pembrolizumab if PD-L1 score high The use of an immune checkpoint inhibitor in this patient was partly based on the approval of pembrolizumab for use in gastric adenocarcinomas for PD-L1 IHC expression ≥1, which was in effect at the time we saw the patient50 but has since been rescinded. There are also data from a pooled meta-analysis of 12 gastric cancer studies, which showed that a CPS ≥1 is the cutoff value for anti–PD-1/anti–PD-L1–based immunotherapy to gain survival benefit.45 Still, it is unclear whether the patient would have responded to pemigatinib alone in the absence of pembrolizumab. CUP is a disease conventionally found in older adults, with a median age of 65 years. This patient was only 36 years old. A retrospective review of young adults diagnosed with CUP at The University of Texas MD Anderson Cancer Center indicated a median age of 35 years, with adenocarcinoma as the predominant histology (70%), and the most common putative primary was biliary tract.51 The prognosis was poor, with median overall survival <1 year. In regard to a possible germline predisposing condition, our patient did not have any significant family history of cancer. Regardless of the patient's family history, genetic counseling and panel testing can be beneficial in this scenario. A positive germline test has the potential to contribute to clinical trial and treatment options (as in the case of a germline BRCA1/BRCA2 or Lynch-related mutation), to inform future cancer risk and screening recommendations for the patient, and to allow for cascade testing of at-risk relatives. It is important to note that somatic testing is not a substitute for germline testing in cases where a clinical suspicion of germline mutation exists because of age at diagnosis or family history.52 Comprehensive germline testing with a 156-gene customized cancer panel (Invitae Inc.; Table 1) revealed no suspicious aberrations in this patient. The treatment paradigm of advanced CUP has remained the same for several years now. Patients with CUP are offered platinum-based and/or paclitaxel-based cytotoxic therapies at most oncology centers in a frontline setting, but the median survival remains 6–15 months.19 Attempts to treat based on tissue-of-origin identification in general have not yielded a survival advantage compared with empiric chemotherapy.53 CUP is almost an ideal tumor for the incorporation of NGS-based therapeutic matching. With this strategy, genomics is the diagnosis.54 Importantly, prior studies have shown that tailored combinations of drugs matched to a majority of the patients' genomic, transcriptomic, and immunomic alterations can be given safely (generally by using initial dose reductions and titrating the doses to tolerance) and that enhanced degrees of matching correlate with improvements in all outcome parameters across multiple tumor types, including CUP.3, 5, 6 Even patients, such as ours, in whom tissue is not available for NGS can have their cancer's genomic status interrogated by evaluating NGS on ctDNA derived from a small vial of blood. Indeed, the incorporation of blood-based NGS has further transformed this field and opened doors to more accessible and less time-consuming diagnostic tools that were almost unimaginable a decade ago Overall, CUP is a heterogenous group of cancers that harbor distinct, characterizable molecular alterations, many of which may be pharmacologically tractable. In the case presented, based on input from experts on our Molecular Tumor Board, a patient with advanced CUP was treated with the FGFR inhibitor pemigatinib and the checkpoint blockade (anti–PD-1 agent) pembrolizumab because her cancer harbored molecular FGFR2 fusion, amplification, and rearrangement and PD-L1 positivity. She has tolerated therapy well and has an ongoing partial response at 7 months. Further prospective trials of NGS-based strategies for CUP are warranted. ■ Aditya V. Shreenivas reports research funding from Natera and serves on the advisory board of Taiho Oncology, all outside the submitted work. Shumei Kato reports contracts with ACT Genomics, Sysmex, Konica Minolta, OmniSeq, and Personalis; speaker's fees from Bayer and Roche; and personal fees from CureMatch, Foundation Medicine, Medpace, NeoGenomics, and Pfizer outside the submitted work. Jason Sicklick reports grants and research funding from Amgen and Foundation Medicine; personal fees from Deciphera; honoraria from Bayer, Deciphera, Foundation Medicine, Hoffman-La Roche, and Merck; membership in the Foundation Medicine, QED Therapeutics, and Roche speakers' bureaus; and has stock and other ownership interests in Personalis, all outside the submitted work. Razelle Kurzrock reports research funding from Biological Dynamics, Boehringer Ingelheim, Debiopharm, Foundation Medicine, Genentech, Grifols, Guardant, Incyte, Konica Minolta, Medimmune, Merck Serono, Omniseq, Pfizer, Sequenom, Takeda, and TopAlliance; personal consulting, speaking, and/or advisory board fees from Actuate Therapeutics, AstraZeneca, Bicara Therapeutics, Biological Dynamics, Daiichi Sankyo Inc., EISAI, EOM Pharmaceuticals, Iylon, Merck, NeoGenomics, Neomed, Pfizer, Prosperdtx, Roche, TD2/Volastra, Turning Point Therapeutics, and X-Biotech; equity interest in CureMatch Inc., CureMetrix, and IDbyDNA; serves on the Boards of CureMatch and CureMetrix; and is a cofounder of CureMatch, all outside the submitted work. Jingjing Hu and Catherine Skefos made no disclosures.